We hypothesize that prostate cancer cells metabolize their arachidonic acid (AA) stores via cyclooxygenases (COX), 5-lipoxygenase (LOX), or 15-LOX-1 to prostaglandin (PG)E2, 5-hydroxy-eicosatetraenoate (5-HETE), or 12-HETE, respectively, that the production of one or more of these eicosanoids is essential for prostate cancer cell proliferation, and that eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) interfere with the oxygenases to inhibit this proliferation. We have developed a model relevant to these hypotheses. Pten[loxp/loxp]xPB-Cre4 (Pten-/-) mice develop prostate lesions that progress from hyperplasia to cancer. This progression is speeded in mice fed an n-6 diet (rich in AA and AA precursors) and suppressed in mice fed an n-3 diet (rich in EPA and DHA). We will test our hypotheses using human and mouse prostate cells in vitro and our animal model in vivo with three Specific Aims. These Aims are designed to show that: 1) Human prostate cancer cell lines and Pten-/-'mouse prostate cells use 5-LOX, COX-1/2, and/or 15-LOX-1 to make 5-HETE, PGE2, and/or 12-HETE to promote their own proliferation in vitro. 2) EPA and DHA interfere with one or more of the oxygenases to block eicosanoid production and thereby inhibit the proliferation of human prostate cancer cell lines and Pten-/- mouse prostate cells in vitro. 3) The n-3 diet similarly interferes with one or more of the oxygenases to block eicosanoid production and the malignant growth of the prostate gland and thereby improves survival in Pten-/- mice. Previous studies have measured AA metabolites in prostate cancer cell lines in vitro but preloaded the cells with AA to detect the metabolites. AA preloading would pervert any investigation into the effects of EPA, DHA, and the n-3 diet on AA metabolism. We have established a reversed-phase high performance liquid chromatography/electrospray ionization tamdem mass spectroscopy method and with it detected for the first time PGE2, 5-HETE, and 12-HETE in resting PC-3 cells not preloaded with AA. This represents a significant advance that will allow us to characterize the metabolites of AA, EPA, and DHA made by prostate cells and tissues in vitro and in vivo. We are therefore positioned to test our hypotheses and thereby to identify the molecular targets and define the mechanism for the anti-cancerous effects of EPA, DHA, and the fish oil diets which contain these fatty acids.